It is sometimes necessary to detect a small amount of a specific biopolymer in a sample which includes a background of a much larger quantity of unrelated material. Some important cases are immunoassays for viruses or anti-pathogen antibodies present at very low levels in blood or other body fluids of infected individuals or for proteins present at very low levels in cell lysates; assays for sparse receptors on cell surfaces; and nucleic acid probe hybridization assays for pathogenic bacteria present at very low levels in food, for pathogenic protozoan parasites, bacteria or viruses present at low levels in body fluids, or for short segments of defined sequence indicative of genetic abnormalities in the total genomic DNA of a species.
The specificity of such an assay depends on the use of an affinity molecule that binds specifically to a biopolymer analyte (i.e., a particular target biopolymer, or a particular target site or segment of a biopolymer) which is present in a sample only if the entity being tested for (e.g., virus, other microogranism, cells with particular receptors, abnormal gene) is present. Examples of affinity molecules include an antibody for a protein target, that is the antigen used to elicit the antibody; an oligonucleotide with a sequence complementary to that of a target segment of a target DNA or RNA; an antigen for an antibody target, which is an antibody elicited by the antigen; and a lectin which binds specifically to a particular carbohydrate moiety of a target glycoprotein or target polysaccharide.
Detection of an affinity molecule, which has bound to any of its biopolymer analyte that is present in a sample being assayed, is achieved typically by complexing with, or including in, the affinity molecule a reporter group which, as part of a reporter system, can produce a detectable signal. The sensitivity of an assay-will depend on both the specificity of binding between the affinity molecule and the biopolymer analyte in assay systems, the specificity of the reporter system in providing signal only from affinity molecule in an assay system, and the intensity of signal generated by the reporter system in an assay system.
Typical reporter systems employ fluorescent organic moieties or .sup.32 P-labeled phosphate groups as reporter groups. The sensitivity that can be achieved with these types of reporter systems, which involve signal directly from reporter groups, is fundamentally limited by the number of reporter groups needed to produce a signal of detectable intensity. This number is about 10.sup.6. Thus with these groups no fewer than about 10.sup.5 target molecules, or target segments, can be detected in an assay.
The sensitivity Of non-radioactive assays for biopolymer analytes has been improved by employing enzyme adducts linked to an affinity molecule. For example, an oligonucleotide or DNA affinity molecule "probe", that is biotinylated, is detected by complexing enzyme-linked avidin reporter group with the biotinyl groups and then detecting product from a reaction catalyzed by the enzyme. Langer et al., Proc. Natl. Acad. Sci. (U.S.A.) 78, 6633-6637 (1981); Leary et al., Proc. Natl. Acad. Sci. (U.S.A.) 80, 4045-4049 (1983). See also, with regard to enzyme-linked avidin as reporter group in immunoassays, Hevey and Malmros, U.S. Pat. No. 4,228,237. By joining an enzyme to the affinity molecule and then providing substrate for a reaction catalyzed by the enzyme, it is possible to accumulate a large number of product molecules for each enzyme-affinity molecule-analyte complex, and hence, in principle, obtain sensitivities much greater than is possible using small reporter molecules directly. Peroxidase and phosphatase, enzymes that are readily assayed by sensitive colorimetric methods, are widely used in enzyme-adduct reporter groups. Leary et al., supra.
However, enzyme-adduct reporter systems, such as that involving phosphatase linked to avidin, are fundamentally limited in sensitivity by the rates at which reactions catalyzed by the enzymes of a reporter group can occur. As a practical matter, a detectable quantity of a product of the enzyme-catalyzed reaction must be produced in an assay within some reasonable time period, between about 1 to about 100 hours. In practice, enzyme-adduct reporter systems are about 10 to 100 times less sensitive than reporter systems based on .sup.32 p-decay.
There exists a need for reporter systems that are more sensitive, even to the extent that the presence of a single molecule of target biopolymer or single target biopolymer segment can, in principle, be detected in an assay that takes no longer than a few hours. Such a reporter system requires that the presence of the analyte be detected through a reaction that produces a prodigious number of product molecules, per analyte molecule or segment, in a relatively short time.
An enzymatically catalyzed, nucleic acid polymerization reaction generates, from a template, a chain of complementary sequence, which is also a substrate for the involved polymerase. Thus, repeat reactions increase the numbers of particular nucleotide chains exponentially. Accordingly, there are advantages in utilizing the self-replicability of nucleic acids to provide sensitivity in reporter systems.
An example of the use of a nucleic acid polymerization reaction to render detectable a minute amount of analyte is provided by R. K. Saiki et al., Science 230, 1350-1354 (1985) and European Patent Application Publication No. 0 164 054. Saiki et al., supra, employ E. coli DNA polymerase I, together with dATP, dCTP, dGTP and dTTP and two synthetic oligonucleotide primers, one with a sequence complementary to a segment near the 3'-end of the sense strand and the other with a sequence complementary to a segment near the 3'-end of the anti-sense strand of the analyte DNA segment, to increase the quantity of analyte in a sample to a level that is readily detectable by standard DNA probe assay techniques. The Saiki et al. procedure involves a number of cycles of DNA polymerase-catalyzed replication, strand-separation, and primer annealing. The amount of analyte increases exponentially with the number of cycles, at least until the concentration of primed segments for replication exceeds the concentration of polymerase molecules. The procedure described in Saiki et al. requires at least three synthetic oligonucleotides, the two primers as well as at least one probe for detection of analyte. Each cycle of replication in the Saiki et al. procedure has three-steps. Thus, while the Saiki et al. procedure enhances sensitivity of a DNA probe assay by exploiting the self-replicability of DNA to increase the amount of analyte, there remains a need for simpler and more generally applicable procedures whereby the sensitivity of-assays for biopolymers or particular segments thereof or sites thereon is enhanced through the self-replicability of nucleic acids.
Enzymatically catalyzed, RNA-directed, RNA polymerizations generate complementary chains rapidly and without need for primers. The numbers of RNA chains increase exponentially with repeated reaction cycles in RNA-directed, RNA polymerizations. Unlike other such polymerizations in vitro, the RNA-directed, RNA polymerizations proceed continuously without need for strand-separation and primer-annealing between cycles. Enzymatically catalyzed, RNA-directed, RNA polymerization has been termed "autocatalytic replication." Haruna and Spiegelman, Science 150, 884-886 (1965). Until the present invention, it has not been appreciated that autocatalytic replication can be employed to provide convenient, broadly applicable, highly sensitive reporter systems for biopolymer analytes.
Miele et al., J. Mol. Biol. 171, 281-295 (1983) describe the insertion of a decaadenylic acid segment into a mutant midivariant-1 RNA at a position which is not essential for function of the RNA-dependent RNA polymerase ("replicase") of bacteriophage Q.beta. and report that the recombinant midivariant-1 RNA remains active as a template for replication by the replicase. See also Kramer et al., U.S. patent application Ser. No. 614,350, filed May 25, 1984, which is incorporated herein by reference. The Kramer et al. application describes recombinant RNA templates, based on midivariant and similar RNAs, for replication by Q.beta. replicase activity and the use of such templates as nucleic acid hybridization probes. Neither the Miele et al. article nor the Kramer et al. patent application suggests the use of a replicative RNA and an associated RNA-dependent RNA polymerase as the basis of a reporter system for assays for biopolymer analytes. Further, neither the article nor the patent application suggests that a recombinant replicative RNA, used as a probe for a target nucleic acid segment, could be replicated subsequent to hybridization with target to increase the sensitivity of assays employing the probe.